Starch is a mixture of amylose, which consists of alpha-1-4 linked glucose, and amylopectin, which comprises alpha-1-4 linked glucose and alpha-1-6 linked glucose. Amylases catalyse the cleavage of glycosidic bonds in amylose, amylopectin, glycogen and related polysaccharides. Specifically, alpha-amylases catalyse the cleavage of hydrolysis of random (1-4)-alpha-D-glucosidic linkages. They belong to family 13 of the CAZY classification of glycosyl hydroxylases.
Amylases and particularly alpha-amylases have been used for different purposes, including cleaning starch-containing stains—particularly in laundry and dishwashing-, textile desizing, starch modification, liquefaction and saccharification—particularly in the pulp & paper industry, for syrup production and in the feed industry-, baking and brewing.
For industrial applications of enzymes, homogeneity of product batches is important. In particular for such applications in which enzymes and enzyme compositions are brought into direct contact with humans or animals, e.g. in cleaning compositions and food or feed production, strict quality control standards have to be employed. One object of such quality control is to ascertain that no adverse substances are accidentally comprised in the composition. Ideally, an enzyme composition should not comprise degraded forms of the respective enzyme which may for example influence the substrate specificity of the enzyme or lead to precipitation. Another object is to ascertain that the enzyme preparation analysed in quality controls is representative for the total lot to be sold. Further, it must be established if and how the enzyme of the enzyme composition will change in the time between sampling for quality control and sales to a customer and/or in the time to final use.
It has now been found that homogeneity of commercially available amylases is to a large scale insufficient. Particularly, it has been unexpectedly found that what is sold as commercial compositions comprising only one enzyme (e.g. Termamyl® or Stainzyme®), i.e. one amylase as ascertained by SDS-PAGE, generally comprises several polypeptide species which can be separated by isoelectric focusing or ion exchange chromatography (herein also termed “parent amylase” and “ghost”/“ghosts”).
Given that protein sequencing is cumbersome and costly, the exact amino acid sequence of polypeptides including enzymes is generally predicted only on the basis of nucleic acid sequences coding for the polypeptides to be produced. However, the above finding that allegedly pure enzyme compositions, i.e. compositions comprising only one species of polypeptide, actually comprise several polypeptide species necessitates the conclusion that the compositions sold comprise significant amounts of polypeptides of unknown primary, secondary and tertiary structure. Thus, unless a complete protein sequencing of all individual amylase species of a composition is performed—which generally is not done-, it is presently not possible to provide enough information to perform a proper freedom-to-operate analysis by amylase manufacturers or customers thereof.